Isolation of crude membranes from animal and plant cells and/or tissues is the first step for further purification and characterization of membrane and organelle proteins. Over 70% of drug targets and biomarkers are found on plasma membranes, therefore isolation and purification of membrane proteins is one of the most commonly employed procedures in biomedical research. Traditional membrane and organelle isolation protocols often involve homogenizing cultured cells and/or tissues in a hypotonic buffer or a buffer containing detergents such as Triton-X 114. The cells and/or tissues are homogenized with a glass homogenizer (such as Dounce homogenizer), sonication or a tissue blender to break up cells and/or tissues. Insoluble membrane and organelles are separated from nuclei and other soluble proteins by differential centrifugations. The procedures are tedious and time consuming; a typical procedure takes more than one hour to complete.
Several commercial kits are available for isolation of membrane proteins. These commercial kits can be roughly classified into two categories based on mechanisms of cell membrane disruption: 1) membrane protein isolation using detergents (for instance, Mem-PER Eukaryotic Membrane Protein Extraction Kit, Pierce, Rockford, Ill.) without using a glass homogenizer and 2) membrane protein isolation using a glass homogenizer (for instance, Plasma Membrane Protein Extraction Kit, Biovison, Mountain View, Calif.), and Plasma Membrane Protein Extraction Kit, abcam, Cambridge, Mass.) or a blender (for instance, Cytosolic and Nuclear Membrane Protein Extraction Protocols, Millipore, Billerica, Mass.) without using detergents.
Methods employing detergents for membrane protein isolation offer the advantages of using smaller amounts of starting cells (at least 5×106 cells) and selectively extracting certain fractions of membrane proteins; however these methods potentially alter the normal configuration of membrane and membrane proteins. Methods using glass homogenizers or blenders for membrane disruption isolate membrane proteins and organelles in their native forms and retain full protein activities. These methods generally require larger amount of starting cells (5×106 and up). The use of glass homogenizer and tissue blenders is generally less user friendly for multiple samples and are time consuming Thorough cleaning of the homogenizer is required for each sample and there is a possibility of sample cross-contamination. Currently available commercial kits for membrane and organelle isolation are relatively tedious and time consuming. In addition to the use of glass homogenizers or tissue blenders, multiple incubation and centrifugation steps are common. These procedures usually take more than one hour to complete. A typical crude membrane isolation protocol in a research laboratory usually requires the following steps: collect cells by low speed centrifugation; wash cells and resuspend in a homogenization buffer with 0.25 M sucrose; break cells in a Dounce homogenizer and check under a phase contract microscope to determine if majority of cells are broken; centrifuge homogenate for 10 minutes at 1700×g; transfer supernatant to a new tube and centrifuge at 33,000×g for 60 minutes; and remove the supernatant leaving a pellet containing crude membranes that include plasma membrane and organelle membranes (Schimmel D. S. et al. (1973). Proc. Nat. Acad. Sci. USA, Vol. 70, pp-3195-3199).